E1 Component Research Articles

Dihydrolipoamide S-acetyltransferase activation alleviates diabetic kidney disease via AMPK-autophagy axis and mitochondrial protection

Diabetic kidney disease (DKD), a severe complication of diabetes marked by deregulated glucose metabolism, remains enigmatic in its pathogenesis. Herein, we delved into the functional role of Dihydrolipoamide S-acetyltransferase (DLAT), a pivotal E2 component of the pyruvate dehydrogenase complex (PDC), in the context of DKD. Our findings revealed a downregulation of DLAT in the kidneys of diabetic patients, correlating inversely with kidney function. Parallel downregulation was observed in both high-fat diet/streptozotocin (HFD/STZ) and db/db mouse models, as well as in human proximal tubular epithelial cells (HK-2) cultured under hyperglycemic conditions. To further elucidate the role of endogenous DLAT in DKD, we employed genetic ablation of Dlat in mouse models. Dlat haploinsufficient mice exhibited exacerbated renal dysfunction, structural damage, fibrosis, and mitochondrial dysfunction under DKD conditions. Consistent with these findings, modulation of DLAT expression in HK-2 cells highlighted its influence on fibrosis, with overexpression attenuating Fibronectin and Collagen I levels, while downregulation exacerbated fibrosis. Mechanistically, we discovered that DLAT activates mitochondria autophagy through the Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway, thereby mitigating mitochondrial dysfunction associated with DKD progression. Inhibition of AMPK abrogated the protective effects of DLAT against mitochondrial dysfunction and DKD. Notably, we identified Hyperforin (HPF), a phytochemical, as a potential therapeutic agent. HPF activates DLAT and AMPK, subsequently ameliorating renal dysfunction, injuries, and fibrosis in both in vivo and in vitro models. In summary, our study underscores the pivotal role of DLAT and AMPK in kidney health and highlights the therapeutic potential of HPF in treating DKD.

Identification of potential biomarkers of cuproptosis in cerebral ischemia.

Cerebral ischemia can cause mild damage to local brain nerves due to hypoxia and even lead to irreversible damage due to neuronal cell death. However, the underlying pathogenesis of this phenomenon remains unclear. This study utilized bioinformatics to explore the role of cuproptosis in cerebral ischemic disease and its associated biomarkers. R software identified the overlap of cerebral ischemia and cuproptosis genes, analyzed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and explored hub genes. Expressions and localizations of hub genes in brain tissue, cells, and immune cells were analyzed, along with predictions of protein structures, miRNAs, and transcription factors. A network was constructed depicting hub gene co-expression with miRNAs and interactions with transcription factors. Ferredoxin 1 (FDX1) expression was determined using qRT-PCR. Ten cuproptosis-related genes in cerebral ischemia were identified, with GO analysis revealing involvement in acetyl-CoA synthesis, metabolism, mitochondrial function, and iron-sulfur cluster binding. KEGG highlighted processes like the tricarboxylic acid cycle, pyruvate metabolism, and glycolysis/gluconeogenesis. Using the Human Protein Atlas, eight hub genes associated with cuproptosis were verified in brain tissues, hippocampus, and AF22 cells. Lipoyl(octanoyl) transferase 1 (LIPT1), was undetected, while others were found in mitochondria or both nucleus and mitochondria. These genes were differentially expressed in immune cells. FDX1, lipoic acid synthetase (LIAS), dihydrolipoamide S-acetyltransferase (DLAT), pyruvate dehydrogenase E1 component subunit alpha 1 (PDHA1), PDHB, and glutaminase (GLS) were predicted to target 111 miRNAs. PDHA1, FDX1, LIPT1, PDHB, LIAS, DLAT, GLS, and dihydrolipoamide dehydrogenase (DLD) were predicted to interact with 11, 10, 10, 9, 8, 7, 5, and 4 transcription factors, respectively. Finally, FDX1 expression was significantly upregulated in the hippocampus of ovariectomized rats with ischemia. This study revealed an association between cerebral ischemic disease and cuproptosis, identifying eight potential target genes. These findings offer new insights into potential biomarkers for the diagnosis, treatment, and prognosis of cerebral ischemia, and provide avenues for the exploration of new medical intervention targets.

UBR1 promotes anaplastic thyroid carcinoma progression via stabilizing YAP through monoubiquitylation

Anaplastic thyroid carcinoma (ATC) is a highly aggressive human malignancy without effective treatment. Yes-associated protein (YAP) is a critical effector of the Hippo pathway, which is essential in thyroid carcinogenesis. However, the underlying mechanisms of aberrant YAP expression in ATC are not completely understood. Ubiquitylation-related enzyme siRNA screening identified the ubiquitin protein ligase E3 component n-recognin 1 (UBR1) as a stabilizer of YAP in ATC cells. UBR1 deficiency reduced YAP protein levels and its target gene expression. UBR1 directly interacted with YAP and promoted its monoubiquitylation, competitively suppressing its polyubiquitylation and resulting in extended protein half-life. UBR1 depletion reduced ATC cell proliferation and migration in vitro. Xenograft tumor studies also suggested that UBR1 knockdown suppressed ATC cell growth in vivo. Furthermore, exogenous YAP expression partially reversed the inhibitive effects of UBR1 depletion on ATC cell proliferation and migration. Our studies demonstrated that UBR1 directly interacts with YAP and stabilized it in a monoubiquitylation-dependent manner, consequently promoting ATC tumorigenesis, suggesting that UBR1 might be a potentially therapeutic target for ATC treatment.

Mitochondrial copper overload promotes renal fibrosis via inhibiting pyruvate dehydrogenase activity

Copper is a trace element essential for numerous biological activities, whereas the mitochondria serve as both major sites of intracellular copper utilization and copper reservoir. Here, we investigated the impact of mitochondrial copper overload on the tricarboxylic acid cycle, renal senescence and fibrosis. We found that copper ion levels are significantly elevated in the mitochondria in fibrotic kidney tissues, which are accompanied by reduced pyruvate dehydrogenase (PDH) activity, mitochondrial dysfunction, cellular senescence and renal fibrosis. Conversely, lowering mitochondrial copper levels effectively restore PDH enzyme activity, improve mitochondrial function, mitigate cellular senescence and renal fibrosis. Mechanically, we found that mitochondrial copper could bind directly to lipoylated dihydrolipoamide acetyltransferase (DLAT), the E2 component of the PDH complex, thereby changing the interaction between the subunits of lipoylated DLAT, inducing lipoylated DLAT protein dimerization, and ultimately inhibiting PDH enzyme activity. Collectively, our study indicates that mitochondrial copper overload could inhibit PDH activity, subsequently leading to mitochondrial dysfunction, cellular senescence and renal fibrosis. Reducing mitochondrial copper overload might therefore serve as a strategy to rescue renal fibrosis.

Methylome-proteome integration after late-life voluntary exercise training reveals regulation and target information for improved skeletal muscle health.

Exercise is a potent stimulus for combatting skeletal muscle ageing. To study the effects of exercise on muscle in a preclinical setting, we developed a combined endurance-resistance training stimulus for mice called progressive weighted wheel running (PoWeR). PoWeR improves molecular, biochemical, cellular and functional characteristics of skeletal muscle and promotes aspects of partial epigenetic reprogramming when performed late in life (22-24months of age). In this investigation, we leveraged pan-mammalian DNA methylome arrays and tandem mass-spectrometry proteomics in skeletal muscle to provide detailed information on late-life PoWeR adaptations in female mice relative to age-matched sedentary controls (n=7-10 per group). Differential CpG methylation at conserved promoter sites was related to transcriptional regulation genes as well as Nr4a3, Hes1 and Hox genes after PoWeR. Using a holistic method of -omics integration called binding and expression target analysis (BETA), methylome changes were associated with upregulated proteins related to global and mitochondrial translation after PoWeR (P=0.03). Specifically, BETA implicated methylation control of ribosomal, mitoribosomal, and mitochondrial complex I protein abundance after training. DNA methylation may also influence LACTB, MIB1 and UBR4 protein induction with exercise - all are mechanistically linked to muscle health. Computational cistrome analysis predicted several transcription factors including MYC as regulators of the exercise trained methylome-proteome landscape, corroborating prior late-life PoWeR transcriptome data. Correlating the proteome to muscle mass and fatigue resistance revealed positive relationships with VPS13A and NPL levels, respectively. Our findings expose differential epigenetic and proteomic adaptations associated with translational regulation after PoWeR that could influence skeletal muscle mass and function in aged mice. KEY POINTS: Late-life combined endurance-resistance exercise training from 22-24months of age in mice is shown to improve molecular, biochemical, cellular and in vivo functional characteristics of skeletal muscle and promote aspects of partial epigenetic reprogramming and epigenetic age mitigation. Integration of DNA CpG 36k methylation arrays using conserved sites (which also contain methylation ageing clock sites) with exploratory proteomics in skeletal muscle extends our prior work and reveals coordinated and widespread regulation of ribosomal, translation initiation, mitochondrial ribosomal (mitoribosomal) and complex I proteins after combined voluntary exercise training in a sizeable cohort of female mice (n=7-10 per group and analysis). Multi-omics integration predicted epigenetic regulation of serine β-lactamase-like protein (LACTB - linked to tumour resistance in muscle), mind bomb 1 (MIB1 - linked to satellite cell and type 2 fibre maintenance) and ubiquitin protein ligase E3 component N-recognin 4 (UBR4 - linked to muscle protein quality control) after training. Computational cistrome analysis identified MYC as a regulator of the late-life training proteome, in agreement with prior transcriptional analyses. Vacuolar protein sorting 13homolog A (VPS13A) was positively correlated to muscle mass, and the glycoprotein/glycolipid associated sialylation enzyme N-acetylneuraminate pyruvate lyase (NPL) was associated to in vivo muscle fatigue resistance.

Multiple E3 ligases act as antiviral factors against SARS-CoV-2 via inducing the ubiquitination and degradation of ORF9b.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) open reading frame 9b (ORF9b) antagonizes the antiviral type I and III interferon (IFN) responses and is ubiquitinated and degraded via the ubiquitin-proteasome pathway. However, E3 ubiquitin ligases that mediate the polyubiquitination and degradation of ORF9b remain unknown. In this study, we identified 14 E3 ligases that specifically bind to SARS-CoV-2 ORF9b. Specifically, three E3 ligases, HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, induced K48-linked polyubiquitination and degradation of ORF9b, thereby attenuating ORF9b-mediated inhibition of the IFN response and SARS-CoV-2 replication. Moreover, each E3 ligase performed this function independent of the other two E3 ligases. Therefore, the three E3 ligases identified in this study as anti-SARS-CoV-2 host factors provide novel molecular insight into the virus-host interaction.IMPORTANCEUbiquitination is an important post-translational modification that regulates multiple biological processes, including viral replication. Identification of E3 ubiquitin ligases that target viral proteins for degradation can provide novel targets for antagonizing viral infections. Here, we identified multiple E3 ligases, including HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, that ubiquitinated and induced the degradation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 9b (ORF9b), an interferon (IFN) antagonist, thereby enhancing IFN production and attenuating SARS-CoV-2 replication. Our study provides new possibilities for drug development targeting the interaction between E3 ligases and ORF9b.

Kv3.1 Interaction with UBR5 Is Required for Chronic Inflammatory Pain.

Chronic inflammatory pain caused by neuronal hyperactivity is a common and refractory disease. Kv3.1, a member of the Kv3 family of voltage-dependent K+ channels, is a major determinant of the ability of neurons to generate high-frequency action potentials. However, little is known about its role in chronic inflammatory pain. Here, we show that although Kv3.1 mRNA expression was unchanged, Kv3.1 protein expression was decreased in the dorsal spinal horn of mice after plantar injection of complete Freund's adjuvant (CFA), a mouse model of inflammatory pain. Upregulating Kv3.1 expression alleviated CFA-induced mechanical allodynia and heat hyperalgesia, whereas downregulating Kv3.1 induced nociception-like behaviors. Additionally, we found that ubiquitin protein ligase E3 component n-recognin 5 (UBR5), a key factor in the initiation of chronic pain, binds directly to Kv3.1 to drive its ubiquitin degradation. Intrathecal injection of the peptide TP-CH-401, a Kv3.1 ubiquitination motif sequence, rescued the decrease in Kv3.1 expression and Kv currents through competitive binding to UBR5, and consequently attenuated mechanical and thermal hypersensitivity. These findings demonstrate a previously unrecognized pathway of Kv3.1 abrogation by UBR5 and indicate that Kv3.1 is critically involved in the regulation of nociceptive behavior. Kv3.1 is thus a promising new target for treating inflammatory pain.

Downstream Target Analysis for miR-365 among Oral Squamous Cell Carcinomas Reveals Differential Associations with Chemoresistance.

Expression of microRNAs, such as miR-365, is known to be dysregulated in many tumors, including oral cancers, although little is known about their role or functions. The objective of this project is to evaluate the downstream targets of miR-365 to determine any potential pathways or effects. Downstream targets for miR-365 (miRdatabase target scores > 90) were used for qPCR screening of oral cancer cell lines (SCC4, SCC9, SCC15, SCC25, CAL27). Each oral cancer cell line expressed miR-365 downstream targets molybdenum cofactor synthesis-2 (MOCS2), erythropoietin receptor (EPOR), IQ motif containing-K (IQCK), carboxypeptidase A3 (CPA3), solute carrier family 24 member-3 (SLC24A3), and coiled-coil domain containing 47 (CCDC47)-although the expression levels varied somewhat. However, differential results were observed with ubiquitin protein ligase E3 component n-recognin-3 (UBR3), nudix hydrolase-12 (NUDT12), zinc finger CCHC-type containing-14 (ZCCHC14), and homeobox and leucine zipper encoding (HOMEZ). These data suggest that many of the miR-365 targets are expressed in the oral cancers screened, with the differential expression of UBR3, ZCCHC14, HOMEZ, and NUDT12, which may be correlated with chemoresistance among two specific oral cancer cell lines (SCC25, SCC9). These results suggest this differential expression may signal potential targets for patient treatment with tumors exhibiting miR-365 and chemotherapeutic resistance.

#2036 Mitochondrial copper overload promotes renal fibrosis via inhibiting pyruvate dehydrogenase activity

Abstract Background and Aims Copper is a trace element essential for numerous biological activities, whereas the mitochondria serve as both major sites of intracellular copper utilization and copper reservoir. Pyruvate dehydrogenase complex (PDHC) is a rate-limiting enzyme of glucose oxidation in mitochondrial matrix, converting pyruvate into acetyl-CoA and linking glycolysis to tricarboxylic acid (TCA) cycle. Dihydrolipoamide acetyltransferase (DLAT), the E2 component of the PDHC, requires lipoylation for enzymatic function and exhibits a high affinity for copper within its lipoyl moiety. Here, we investigated the impact of mitochondrial copper overload on the tricarboxylic acid cycle and renal senescence and fibrosis. Method Molecular dynamics simulation was used to explore the effect of copper ions on lipoylated DLAT protein. ICP-MS was used to detect copper content in mitochondria. Western blotting was used to detect the level of lipoylated DLAT dimer. Enzyme biopsy test kit, electron microscopy, mitoSOX Red staining and ATP content detection were used to evaluate mitochondrial function. Detect Collagen I, α-smooth muscle actin (α-SMA) expression levels and Masson staining to explore renal fibrosis. To reduce mitochondrial copper level, we used lentivirus to downregulate CRT1 expression or copper chelator tetrathiomolybdate in renal tubular epithelial cells (NRK-52E), and used CTR1 knockdown transgenic mice or tetrathiomolybdate in vivo. Results We found that copper ion levels are significantly elevated in the mitochondria in fibrotic kidney tissues, which are accompanied by reduced pyruvate dehydrogenase (PDH) activity, mitochondrial dysfunction, cellular senescence and renal fibrosis. Conversely, lowering mitochondrial copper levels through knocking-down of copper transporter 1 or treatment with the copper chelator tetrathiomolybdate effectively restore PDH enzyme activity, improve mitochondrial function, and mitigate cellular senescence and renal fibrosis. Mechanically, we found mitochondrial copper binds directly to lipoylated dihydrolipoamide acetyltransferase (DLAT), thereby changing the interaction between protein subunits, inducing lipoylated DLAT protein dimerization, and ultimately inhibiting PDH enzyme activity. Conclusion Our study indicates that mitochondrial copper overload could inhibit PDH activity, subsequently leading to mitochondrial dysfunction, cellular senescence and renal fibrosis. Reducing mitochondrial copper overload might therefore serve as a strategy to rescue renal fibrosis.

A review: targeting UBR5 domains to mediate emerging roles and mechanisms - chance or necessity?

Ubiquitinases are known to catalyze ubiquitin chains on target proteins to regulate various physiological functions like cell proliferation, autophagy, apoptosis, and cell cycle progression. As a member of E3 ligase, ubiquitin protein ligase E3 component n-recognin 5 (UBR5) belongs to the HECT E3 ligase and has been reported to be correlated with various pathophysiological processes. In this review, the authors give a comprehensive insight into the structure and function of UBR5. The authors discuss the specific domains of UBR5 and explore their biological functions separately. Furthermore, the authors describe the involvement of UBR5 in different pathophysiological conditions, including immune response, virus infection, DNA damage response, and protein quality control. Moreover, the authors provide a thorough summary of the important roles and regulatory mechanisms of UBR5 in cancers and other diseases. On the whole, investigating the domains and functions of UBR5, elucidating the underlying mechanisms of UBR5 with various substrates in detail may provide new theoretical basis for the treatment of diseases, including cancers, which could improve future studies to construct novel UBR5-targeted therapy strategies.

VIRMA promotes the progression of head and neck squamous cell carcinoma by regulating UBR5 mRNA and m6A levels.

Head and neck squamous cell carcinoma (HNSCC) is a globally prevalent and lethal cancer form which precise mechanisms remain incompletely understood. Increasing evidence suggests that N6-methyladenosine (m6A) plays a crucial role in cancer progression. This study aimed to explore the biological function of m6A modification and vir-like m6A methyltransferase associated (VIRMA) in HNSCC. We conducted an analysis of VIRMA expression in HNSCC cells using The Cancer Genome Atlas (TCGA) database and employed reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting to assess its expression levels in HNSCC cell lines. Additionally, m6A levels in HNSCC cells were quantified, and the correlation between VIRMA expression levels and the clinical and pathological features of other genes was analyzed. Upon knocking down VIRMA levels, we assessed HNSCC cell proliferation, migration, and invasion and validated downstream genes using RT-qPCR and western blot. Our findings suggested that VIRMA, as an m6A-related regulator, may significantly influence HNSCC progression by regulating ubiquitin protein ligase E3 component N-recognin 5 (UBR5) through m6A modification. Therefore, VIRMA may serve as a prognostic biomarker.

Mitochondrial remodeling underlying age-induced skeletal muscle wasting: let’s talk about sex

Sarcopenia is associated with reduced quality of life and premature mortality. The sex disparities in the processes underlying sarcopenia pathogenesis, which include mitochondrial dysfunction, are ill-understood and can be decisive for the optimization of sarcopenia-related interventions. To improve the knowledge regarding the sex differences in skeletal muscle aging, the gastrocnemius muscle of young and old female and male rats was analyzed with a focus on mitochondrial remodeling through the proteome profiling of mitochondria-enriched fractions. To the best of our knowledge, this is the first study analyzing sex differences in skeletal muscle mitochondrial proteome remodeling. Data demonstrated that age induced skeletal muscle atrophy and fibrosis in both sexes. In females, however, this adverse skeletal muscle remodeling was more accentuated than in males and might be attributed to an age-related reduction of 17beta-estradiol signaling through its estrogen receptor alpha located in mitochondria. The females-specific mitochondrial remodeling encompassed increased abundance of proteins involved in fatty acid oxidation, decreased abundance of the complexes subunits, and enhanced proneness to oxidative posttranslational modifications. This conceivable accretion of damaged mitochondria in old females might be ascribed to low levels of Parkin, a key mediator of mitophagy. Despite skeletal muscle atrophy and fibrosis, males maintained their testosterone levels throughout aging, as well as their androgen receptor content, and the age-induced mitochondrial remodeling was limited to increased abundance of pyruvate dehydrogenase E1 component subunit beta and electron transfer flavoprotein subunit beta. Herein, for the first time, it was demonstrated that age affects more severely the skeletal muscle mitochondrial proteome of females, reinforcing the necessity of sex-personalized approaches towards sarcopenia management, and the inevitability of the assessment of mitochondrion-related therapeutics.

NAP1L1 regulates BIRC2 ubiquitination modification via E3 ubiquitin ligase UBR4 and hence determines hepatocellular carcinoma progression

We have previously shown that nucleosome assembly protein 1-like 1 (NAP1L1) plays an important role in the abnormal proliferation of hepatocellular carcinoma (HCC) cells. However, the effects of NAP1L1 on the malignant behaviour of HCC cells, including cell migration, invasion and apoptosis, remain unclear. Baculoviral IAP repeat-containing 2 (BIRC2) plays a key role in initiating the abnormal proliferation, apoptotic escape and multidrug resistance of HCC cells; however, the mechanisms through which its stability is regulated in HCC remain elusive. Here, we found that knockdown of NAP1L1 inhibited the proliferation of HCC cells and activated apoptotic pathways but did not remarkably affect the migratory and invasive abilities of HCC cells. In addition, knockdown of NAP1L1 did not alter the expression of BIRC2 at the transcriptional level but substantially reduced its expression at the translational level, suggesting that NAP1L1 is involved in the post-translational modification (such as ubiquitination) of BIRC2. Furthermore, BIRC2 was highly expressed in human HCC tissues and promoted the proliferation and apoptotic escape of HCC cells. Co-immunoprecipitation (Co-IP) assay and mass spectrometry revealed that NAP1L1 and BIRC2 did not bind to each other; however, ubiquitin protein ligase E3 component n-recognin 4 (UBR4) was identified as an intermediate molecule associating NAP1L1 with BIRC2. Knockdown of NAP1L1 promoted the ubiquitin-mediated degradation of BIRC2 through the ubiquitin–protein junction of UBR4, which in turn inhibited the proliferation and apoptotic escape of HCC cells and exerted anti-tumour effects. In conclusion, this study reveals a novel mechanism through which NAP1L1 regulates the ubiquitination of BIRC2 through UBR4, thereby determining the progression of HCC. Based on this mechanism, suppression of NAP1L1 may inhibit tumour progression in patients with HCC with high protein expression of NAP1L1 or BIRC2.

Rapid screening of point mutations by mismatch amplification mutation assay PCR

Metabolic engineering frequently makes use of point mutation and saturation mutation library creation. At present, sequencing is the only reliable and direct technique to detect point mutation and screen saturation mutation library. In this study, mismatch amplification mutation assay (MAMA) PCR was used to detect point mutation and screen saturation mutation library. In order to fine-tune the expression of odhA encoding 2-oxoglutarate dehydrogenase E1 component, a saturating mutant library of the RBS of odhA was created in Corynebacterium glutamicum P12 based on the CRISPR-Cas2a genome editing system, which increased the l-proline production by 81.3%. MAMA PCR was used to filter out 42% of the non-mutant transformants in the mutant library, which effectively reduced the workload of the subsequent fermentation test and the number of sequenced samples. The rapid and sensitive MAMA-PCR method established in this study provides a general strategy for detecting point mutations and improving the efficiency of mutation library screening.Key points• MAMA PCR was optimized and developed to detect point mutation.• MAMA PCR greatly improves the screening efficiency of point mutation.• Attenuation of odhA expression in P12 effectively improves proline production.

UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

The Retinoic acid-Inducible Gene I (RIG-I) like receptors (RLRs) are the major viral RNA sensors essential for the initiation of antiviral immune responses. RLRs are subjected to stringent transcriptional and posttranslational regulations, of which ubiquitination is one of the most important. However, the role of ubiquitination in RLR transcription is unknown. Here, we screen 375 definite ubiquitin ligase knockout cell lines and identify Ubiquitin Protein Ligase E3 Component N-Recognin 5 (UBR5) as a positive regulator of RLR transcription. UBR5 deficiency reduces antiviral immune responses to RNA viruses, while increases viral replication in primary cells and mice. Ubr5 knockout mice are more susceptible to lethal RNA virus infection than wild type littermates. Mechanistically, UBR5 mediates the Lysine 63-linked ubiquitination of Tripartite Motif Protein 28 (TRIM28), an epigenetic repressor of RLRs. This modification prevents intramolecular SUMOylation of TRIM28, thus disengages the TRIM28-imposed brake on RLR transcription. In sum, UBR5 enables rapid upregulation of RLR expression to boost antiviral immune responses by ubiquitinating and de-SUMOylating TRIM28.

Identification and expression analysis of apple PDHB-1 gene family

Pyruvate dehydrogenase E1 component subunit beta-1 (PDHB-1) is a gene encoding the β-subunit of pyruvate dehydrogenase complex, which plays an important role in fruit acid accumulation. The aim of this study was to investigate the evolution characteristics of apple PDHB-1 family and its expression in apples with different acid contents. Bioinformatics analysis was performed using databases including NCBI, Pfam and software including ClustalX, MEGA, and TBtools. By combining titratable acid content determination and quantitative real-time PCR (qRT-PCR), the expression of this family genes in the peel and pulp of apple 'Asda' and 'Chengji No.1' with different acid content were obtained, respectively. The family members were mainly located in chloroplast, cytoplasm and mitochondria. α-helix and random coil were the main factors for the formation of secondary structure in this family. Tissue-specific expression profiles showed that the expression of most members were higher in fruit than in other tissues. qRT-PCR results showed that the expression profile of most members was consistent with the profile of titratable acid contents. In the peel, the expression levels of 14 members in 'Asda' apples with high acid content were significantly higher than that in 'Chengji No.1' apples with low acid content, where the expression difference of MdPDHB1-15 was the most significant. In the pulp, the expression levels of 17 members in 'Asda' apples were significantly higher than that in 'Chengji No.1' apples, where MdPDHB1-01 was the most highly expressed. It was predicted that PDHB-1 gene family in apple plays an important role in the regulation of fruit acidity.

LncRNA OIP5-AS1 Targets the miR-140-5p/UBR5 Cascade to Promote the Development of Gastric Cancer.

Gastric cancer (GC) is a malignant tumor with the highest incidence among all kinds of malignant tumors in China. Long noncoding RNAs (LncRNAs) have been reported to act as microRNA (miRNAs) sponges and thus play key roles in biological processes and pathogenesis. Thus, this study aimed to investigate the functional effects and the regulatory mechanism of lncRNA opa interacting protein 5-antisense 1 (OIP5-AS1) in gastric cancer cells. The expression of OIP5-AS1, miR-140-5p, Ubiquitin protein ligase E3 component n-recognin 5 (UBR5) was detected using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, apoptosis, migration, and invasion were assessed using Cell-Counting Kit-8 (CCK-8), Flow cytometry, and Transwell assays. UBR5 protein level was detected by Western blot. Binding between miR-140-5p and OIP5-AS1 or UBR5 was predicted by Starbasev2.0 and TargetScan, and verified using Dual-luciferase reporter assays and RNA pull-down assay. A xenograft mice model was used to evaluate the effects of OIP5-AS1 on tumor growth in vivo. OIP5-AS1 was upregulated in GC cancer and cells. OIP5-AS1 knockdown inhibited cell proliferation, migration, invasion, but induced cell apoptosis in GC. In mechanism, OIP5-AS1 might serve as a sponge for miR-140-5p to enhance UBR5 expression. Moreover, overexpression of miR-140-5p or UBR5 partly reversed the effects of OIP5-AS1 depletion on the progression of GC cells. Furthermore, OIP5-AS1 depletion also suppressed tumor growth in vivo. OIP5-AS1 silencing might suppress proliferation, migration, invasion, and induced apoptosis in GC cells by regulating the miR-140-5p/UBR5 axis.

EGR1-mediated metabolic reprogramming to oxidative phosphorylation contributes to ibrutinib resistance in B-cell lymphoma

AbstractThe use of Bruton tyrosine kinase inhibitors, such as ibrutinib, to block B-cell receptor signaling has achieved a remarkable clinical response in several B-cell malignancies, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Acquired drug resistance, however, is significant and affects the long-term survival of these patients. Here, we demonstrate that the transcription factor early growth response gene 1 (EGR1) is involved in ibrutinib resistance. We found that EGR1 expression is elevated in ibrutinib-resistant activated B-cell–like subtype DLBCL and MCL cells and can be further upregulated upon ibrutinib treatment. Genetic and pharmacological analyses revealed that overexpressed EGR1 mediates ibrutinib resistance. Mechanistically, TCF4 and EGR1 self-regulation induce EGR1 overexpression that mediates metabolic reprogramming to oxidative phosphorylation (OXPHOS) through the transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation increases intracellular adenosine triphosphate production, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with metformin or IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting EGR1-mediated metabolic reprogramming to OXPHOS with metformin or IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.

Metabolic engineering of fast-growing Vibrio natriegens for efficient pyruvate production

BackgroundPyruvate is a widely used value-added chemical which also serves as a hub of various metabolic pathways. The fastest-growing bacterium Vibrio natriegens is a promising chassis for synthetic biology applications with high substrate uptake rates. The aim of this study was to investigate if the high substrate uptake rates of V. natriegens enable pyruvate production at high productivities.ResultsTwo prophage gene clusters and several essential genes for the biosynthesis of byproducts were first deleted. In order to promote pyruvate accumulation, the key gene aceE encoding pyruvate dehydrogenase complex E1 component was down-regulated to reduce the carbon flux into the tricarboxylic acid cycle. Afterwards, the expression of ppc gene encoding phosphoenolpyruvate carboxylase was fine-tuned to balance the cell growth and pyruvate synthesis. The resulting strain PYR32 was able to produce 54.22 g/L pyruvate from glucose within 16 h, with a yield of 1.17 mol/mol and an average productivity of 3.39 g/L/h. In addition, this strain was also able to efficiently convert sucrose or gluconate into pyruvate at high titers.ConclusionA novel strain of V. natriegens was engineered which was capable to provide higher productivity in pyruvate synthesis. This study lays the foundation for the biosynthesis of pyruvate and its derivatives in fast-growing V. natriegens.

Double-edged effect of sodium citrate in Nile tilapia (Oreochromis niloticus): Promoting lipid and protein deposition vs. causing hyperglycemia and insulin resistance.

Citrate is an essential substrate for energy metabolism that plays critical roles in regulating glucose and lipid metabolic homeostasis. However, the action of citrate in regulating nutrient metabolism in fish remains poorly understood. Here, we investigated the effects of dietary sodium citrate on growth performance and systematic energy metabolism in juvenile Nile tilapia (Oreochromis niloticus). A total of 270 Nile tilapia (2.81±0.01g) were randomly divided into three groups (3 replicates per group, 30 fish per replicate) and fed with control diet (35% protein and 6% lipid), 2% and 4% sodium citrate diets, respectively, for 8 weeks. The results showed that sodium citrate exhibited no effect on growth performance (P>0.05). The whole-body crude protein, serum triglyceride and hepatic glycogen contents were significantly increased in the 4% sodium citrate group (P < 0.05), but not in the 2% sodium citrate group (P>0.05). The 4% sodium citrate treatment significantly increased the serum glucose and insulin levels at the end of feeding trial and also in the glucose tolerance test (P < 0.05). The 4% sodium citrate significantly enhanced the hepatic phosphofructokinase activity and inhibited the expression of pyruvate dehydrogenase kinase isozyme 2 and phosphor-pyruvate dehydrogenase E1 component subunit alpha proteins (P < 0.05). Additionally, the 4% sodium citrate significantly increased hepatic triglyceride and acetyl-CoA levels, while the expressions of carnitine palmitoyl transferase 1a protein were significantly down-regulated by the 4% sodium citrate (P < 0.05). Besides, the 4% sodium citrate induced crude protein deposition in muscle by activating mTOR signaling and inhibiting AMPK signaling (P < 0.05). Furthermore, the 4% sodium citrate significantly suppressed serum aspartate aminotransferase and alanine aminotransferase activities, along with the lowered expression of pro-inflammatory genes, such as nfκb, tnfα and il8 (P < 0.05). Although the 4% sodium citrate significantly increased phosphor-nuclear factor-kB p65 protein expression (P < 0.05), no significant tissue damage or inflammation occurred. Taken together, dietary supplementation of sodium citrate could exhibit a double-edged effect in Nile tilapia, with the positive aspect in promoting nutrient deposition and the negative aspect in causing hyperglycemia and insulin resistance.